title: Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.
abstract: The combination of laser capture microdissection (LCM) and gene expression experiments allows cell specific expression profiling, which is decisive in cellular transcriptomic exploration. LCM makes possible the isolation of unique cells or group of cells, but maintaining RNA quality during this process is challenging. Several protocols are available for section preparation, but none of those guarantees the integrity of the RNA during microdissection, and operators are recommended to perform LCM during a limited time. We hypothesized that the cause of RNA degradation during the microdissection time is the presence of water rendering endogenous RNase activity possible. We thus developed two methods that stabilize RNA during microdissection time for up to 90 min. The first one consists of performing LCM under an argon atmosphere, thus preventing tissue rehydration; it is compliant with all existing microdissection protocols. The second one is a new fixation and staining method using ethanol as solvent in all preparatory steps to LCM that enhances fixation and dehydration of samples. We assessed several stains in regard of their effect on tissue morphology and RNA integrity and adjusted an ethanolic staining solution of cresyl violet and eosin Y.
authors.affiliation: INSERM U781, Faculté de Médecine, Hôpital Necker-Enfants Malades, Université René-Descartes, 75015, Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Clément-Ziza
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.forename: Francis
authors.initials: F
authors.lastname: Jaubert
authors.forename: Claude
authors.initials: C
authors.lastname: Besmond
mesh_terms: D001128:Argon / Q000737:chemistry*; D003106:Colon / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D020871:RNA Stability
chemical_list: D000431:Ethanol; D012313:RNA; D001128:Argon